Changing trends in antibiotic susceptibility pattern among clinical isolates of Pseudomonas species in a tertiary care hospital in Nepal

RESULTS A total of 42,545 specimens comprising of sputum, blood, pus, urine, body fluids and endotracheal tubes were processed during the entire 3 year period. There was significant increase (p-value <0.05) in yearly isolation rate of Pseudomonas spp i.e. 141(7.95%), 197 (12.14%) and 303(15.69%) isolates in the year 2017, 2018, and 2019 respectively. Resistance rates of the isolates to cefoparazone sulbactam, Piperacillin Tazobactam, and Tobramycin was significantly decreasing (p-value <0.05). Overall resistance to ceftazidime (66.13%) was alarming. Decrease in MDR isolates were statistically significant over 3 years e.g. 32 (22.69%), 31(15.73%) and 37(12.21%) isolates in the year 2017, 2018 and 2019 respectively (p-value <0.05).


INTRODUCTION
Pseudomonas species, member of the family Pseudomonadaceae, is an aerobic, gram negative bacilli. It is saprophyte, commonly found in soil, and water, but have emerged as important human pathogen. Pseudomonas spp is a notorious nosocomial pathogen 1 . It is gaining importance as a cause of community acquired pneumonia, blood stream infection, wound infections, including those of burn wounds. Pathogenicity of Pseudomonas is associated with numerous virulence factors like liposaccharide, type VI secretion system, biofilm production, exotoxin A, proteases and alginate production, etc. 2 .
Antibiotic therapy of Pseudomonas infections is challenging as these are intrinsically resistant to a number of antibiotics, carry genes for antibiotic resistance and have adapted multiple mechanisms of antibiotic resistance 3 . Even, resistance among Pseudomonas spp to carbapenem, the last resort antibibiotic, is on steady increase. This trait of intrinsic and acquired antibiotic resistance has narrowed down the choice of antibiotics and clinician has to rely on more toxic and more expensive antibiotics. In context to Nepal, authors like Baral et al, Yadav et al have reported MDR Pseudomonas as high as 60.1%, 73.3% respectively and are associated with prolonged hospital stay and increased mortality which is a serious situation 4,5 . Such emergence of multidrug resistance (MDR), and carbepenem resistance are of primary concern globally, as these factors are importantly related to prolonged hospital stay, increased morbidity and mortality 6 .
The rate of infection by Pseudomonas spp vary in different clinical settings. There is a need of proper surveillance on occurrence of infection by Pseudomonas and their pattern of antibiotic susceptibility. This will guide clinicians to choose the best antibiotics for empirical therapy without delay. This study was, therefore, aimed to find out the infection rate due to Pseudomonas spp and determine the changing trend in antibiotic resistance pattern among the isolates.

Setting and Design
This is an observational and descriptive study conducted at the tertiary care hospital located in Nepal. The culture and sensitivity data of non-repeating isolates of Pseudomonas spp for 3 consecutive years (2017, 2018 and 2019) formed the material of the study.

Isolation and identification of Organism
All the clinical samples obtained in microbiological laboratory were processed by standard microbiological techniques. The specimens had been cultured on Chocolate agar, 5% Sheep Blood agar, MacConkey agar plates and biphasic media (Hi-Media, India) based on type of clinical samples. Isolates were identified as Pseudomonas spp by standard, conventional phenotypic methods using Triple sugar iron agar(TSI), Sulphur Indole Motility agar(SIM), Christensen's urease agar, Simmons citrate agar, oxidase strip and pigment production 7 .

Statistical Analysis
Data were analyzed by SPSS version 20. Categorical variables were calculated as percentages. Chi-square test was used to compare two groups. All p values <0.05 were considered as statistically significant.

RESULTS
A total of 42,545 specimens comprising of sputum, blood, pus, urine, body fluids and endotracheal tubes were processed during the entire three year period. Of these, 13675 specimens were processed in the year 2017, 13780 specimens in 2018, and 15090 in 2019, which yielded 1772, 1622 and 1930 gram negative bacilli respectively. Out of these, Pseudomonas spp accounted for 141(7.95%), isolates in the year 2017, 197(12.14%) isolates in 2018, and 303(15.69%) in 2019. The isolation rate of Pseudomonas spp was found to have significantly increased (p-value <0.05) over the years. Distribution of Pseudomonas spp according to gender, age and season is shown in table no 1. It was found that, in all 3 years, numbers of males suffering from Pseudomonas infection were significantly higher compared to females. Pseudomonas spp was isolated from subjects belonging to all age groups starting from neonates to individuals as old as or even older than 80 years. It was noticed that majority of Pseudomonas i.e. 47.42% (304/641) were isolated from adults (15-60 years of age), while 194(30.26%) and 143(22.30%) were from individuals of old age group (>60 years) and those of younger age group (0-14 years) respectively.
Pseudomonas spp isolation rate was more during the rainy season i.e. 371(57.8%) when compared with other months 270 (42.12%) (p-value >0.05).   The outcome of infections is also associated with increased morbidity and mortality.

Year of isolation
In this study, increase in number of Pseudomonas spp infection in consecutive years was observed. In all 3 years, total number of males affected were 19,754 and females were 21,228. Out of these, number of males whose specimens yielded Pseudomonas on culture were 385, a figure that was higher than the comparable figure among the females (n=256). Though this overall difference was not a significant one (p-value >0.05), yet, it was an interesting observation of male predominance of getting disease. Yadav et al have documented similar observation of male patients getting more Pseudomonas spp infections when compared to females 5 . The reason could be multifactorial but the important factor could be males were found to be less compliant to hand hygiene practice in terms of timing, use of soap, and hand washing behavior as compared to females 11,12 . Moreover, other factors like cultural, hormonal, immune status could also be related to male predominance of not only getting the disease, but also landing up with poor clinical outcome as well 13 .
In the present study, Pseudomonas spp was isolated starting from neonates to individuals as old as or >80 years of age. It was observed that majority of Pseudomonas spp (304; 47.42%) were obtained from patients of the adult age group, whereas there were comparatively lesser numbers (194; 30.26%) from those belonging to the old age group. In spite of the fact that, the older age group category needs to be managed with optimum care as these individuals are often vulnerable to other co-morbid conditions and are prone to infections as a result of seasonal climatic variations 14 . In Pokhara, rainy season extends from May to October 15 . As, Pseudomonas spp is a saprophytic organism, its dissemination is expected to be more in rainy season causing high number of infectious episodes 16 . In our study, 24,298 cultures were performed in rainy season and 17,355 during the non-rainy season. It was of note that Pseudomonas spp isolation rate was higher in the rainy months i.e. 371(57.8%) when compared to the dry parts of the year (270; 42.12%). Though this difference was not statistically significant, (p-value >0.05), yet it was a notable difference which might have implications for the population in the community, especially for those in the old and much older age group.
Antibiotic resistance is the most feared aspect of bacterial infections. WHO warned it as a serious global situation. It was interesting and good to note that the dominance of antibiotic resistant Pseudomonas spp was in a decreasing trend ( Table 2). Even the Multidrug resistant pseudomonas was on a decreasing trend (pvalue <0.05), as shown by the present study. This declining trend in antibiotic resistance exhibited a statistically significant difference (pvalue <0.05), especially with respect to antibiotics, such as cefoparazone sulbactam, piperacillin-tazobactam, tobramycin (table 2). This observation of ours was amply supported by the findings shown elsewhere 17 .
However, resistance rates seen in our study against ceftazidime, an antipseudomonal cephalosporin, was quite high varying between 58.33% in 2017 to 62.07% in 2018. This seems to be alarming. According to recent studies from Nepal as well as from other countries, cephalosporins were one of the most widely used antibiotics for many deep seated infections and other infections, as clinician prescribed and as self-used antibiotic 18,19 .
Thus, our results could possibly implicate the rational use of cephalosporins in order to avoid rapid development of resistance to this drug by notorious bugs like Pseudomonas. Quinolone use and development of multidrug resistance were previously found to be interrelated 20 . In this context, it is important to note that ciprofloxacin is increasingly being used as a prescribed as well as a self-medicated antibiotic 19,21 . Beside these, resistance to β-lactam antibiotics is governed by common mechanism ie β-lactamase production which can be trans-  24,25 . Despite their systemic toxicity colistin and polymyxin B are regarded as the ultimate therapeutic option for critically ill patients due to gram negative bacterial infection 26 .
As per the present data, less than 20% Pseudomonas spp were found to be resistant to ciprofloxacin and aminoglycosides like gentamicin, tobramycin, amikacin. Amikacin was found best choice with resistance rate 11.87%. The resistance rate of these antibiotics has decreased in 2019 when compared with 2017 suggesting these WHO watch group of antibiotics are still better option for management of infection by Pseudomonas spp.
Our antibiotic susceptibility pattern of Pseudomonas isolates shows increase in susceptibility in recent years to plenty of antibiotics tested. Such a trend is favorable. In our study, factors that might have played the role in the increase in susceptibility pattern to certain antibiotics were not looked into. Even then the important observations we made certainly pave the way for further research.